1. Field of the Invention
The present invention relates to colony stimulating factor (CSF)-gelatin conjugates, and processes for the production and use of the conjugates. The conjugates include cross-linked gelatin microspheres and water-soluble CSF-gelatin conjugates. Both conjugates are pharmaceutical forms useful as a tumor growth inhibitor.
2. Description of the Related Art
CSFs are substances which act on mammalian myeloid leucocyte precursor cells to promote their differentiation to granulocytes or macrophages, and which are necessary for differentiation and growth of the myeloid leucocyte precursor cells, which result in the formation of colonies comprising of neutrophil granulocytes (abbreviated as granulocyte hereinafter) or monocytes/macrophages, when the precursor cells are cultured by a two-layer agar culture method (Ichikawa, Y. et al., Proceedings of the National Academy of Science, 56, p488, 1966; Metcalf, D., Experimental Hematology 1, p185, 1972). It is known that CSFs compose of various types possessing different activities, including granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), and multifunctional CSF (multi CSF or IL-3). Considering their functions for the differentiation and growth of leucocyte, CSFs are promising as therapeutic drugs for the treatment of leucopenia caused by the application of anticancer agents or radiation. Moreover, CSF activates mature macrophage to manifest antitumor activity (Weinberg, J.B. et al., Journal of Immunology, 121, p72, 1978; Ralph, P and Nakoing I., Cellular Immunology, 105, p270, 1978). CSFs are also promising for the treatment of cancer, and for the prophylaxis and therapy of infections caused by viruses, bacteria, and parasites because of their functions as macrophage activating factors.
However, the activity of CSFs to activate macrophages is low, and they themselves are unstable and readily degraded in vivo. Therefore, it is of prime importance to develop administration forms of CSFs aiming at an enhanced macrophage activating function and an increased stability in vivo of CSFs.
Japanese Unexamined Patent Publication (KOKAI) No. 59-46215 describes gelatin microcapsules intended for in vivo stabilization of an active component and its sustained release. However, the average diameters of microcapsules is ranging from 10 to 500 .mu.m, and too large to be phagocytized by macrophages. Moreover, no description on microcapsules containing CSF is disclosed. Japanese Unexamined Patent Publication No. 62-230729 describes a pharmaceutical form composed of gelatin to gradually release CSF. However, since the form is produced by grinding gelatin blocks including CSF to fine powders, the resulting powders are not particles small enough to be phagocytized by macrophages. Moreover, since gelatin has not been cross-linked according to this method, and the in vitro degradation of the powders is rapid, the advantages provided by the present microspheres are not provided by the powders. Japanese Unexamined Patent Publication No. 62-143974 describes polymer microspheres containing immunopotentiators. But, the immunopotentiators used are water-soluble muramyldipeptides (MDP), their lipophilic derivatives, krestin, Nocardia rubra, interferons, lipopolysaccharides (LPS), tumor necrosis factor, pyrane copolymer, lymphokines, BCG, and the like, and do not include CSFs. In addition, the publication No. 62 -143974 does not suggest a combination of the crosslinked gelatin with CSF and the combination is a new description. Y. Tabata et al., Jpn. J. Cancer Res. (Gann), 79, p636-646, 1988 describes gelatin microspheres containing interferons, but do not suggest the application of the disclosed techniques to CSF.
Generally, physiologically bioactive substances possess individual physiological activities and action mechanisms to living systems. Therefore, even if it is known that gelatin microspheres containing a physiologically bioactive substance can be phagocytized by macrophages, resulting in the appearance of its physiological action, this does not directly mean that another physiologically bioactive substance processed by the same procedure will successfully provide a similar result. Accordingly, even though it is known that gelatin microspheres containing interferon exhibit its activities through their phagocytosis by macrophages, this does not necessarily suggest that the application of the same procedure to CSF will provide the same results.